Partial purification, and further characterization, of kievitone hydratase from cell-free culture filtrates of Fusarium solani f. sp. phaseoli.

Abstract

Kievitone hydratase (KHase), an enzyme which catalyses detoxification, of the phytoalexin kievitone, was isolated from cell-free culture filtrates of Fusarium solani f. sp. phaseoli . Partial purification was achieved by anion exchange chromatography, gel filtration and gradient Polyacrylamide disc gel electrophoresis. Gel filtration and electrophoresis gave molecular weight estimates of 173 000 and 102 000, respectively. Based on the results of isoelectric focusing and affinity chromatography, KHase is an acidic (pI= 5·1±0·6) glycoprotein. Most enzymic activity was retained after exposure of KHase to sodium periodate oxidation for 24 h. Further characterization revealed that the enzyme had a temperature optimum of 55°C, but denatured rapidly at or above 70°C. The pH optimum was 5·5 at 27°C with an apparent K m of 1·75×10 −6 M under these conditions. Preliminary findings indicated that KHase occurred in Fusarium -infected bean tissues.